Throughout this application various publications are referenced by Arabic numerals in brackets. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are in their entirety hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Herpesviruses contain 100,000 to 200,000 base pairs of DNA as their genetic material having a long unique segment and a short unique segment, the short unique segment bounded by an internal repeat sequence and a terminal repeat sequence. Within a given genome, several regions have been identified that are not essential for the replication of virus in cell culture. Modifications of some of these regions of the DNA have been known to lower the pathogenicity of the virus, i.e., to attenuate the virus when it infects an animal species. For example, inactivation of the thymidine kinase gene of either human herpes simplex virus [1] or pseudorabies virus of swine [2] renders the human herpesvirus less pathogenic in mice and the pseudorabies virus less pathogenic in swine.
Removal of specific regions of the repeat region of a human herpes simplex virus has been shown to render the virus less pathogenic [1, 3]. Furthermore, a repeat region has been identified in Marek""s disease virus that is associated with viral oncogenicity [4]. A region in herpesvirus saimiri has similarly been correlated with oncogenicity [5]. Removal of a specific region of the repeat region renders pseudorabies virus less pathogenic (U.S. Pat. No. 4,877,737). A region in pseudorabies virus has been shown to be deleted in naturally-occurring vaccine strains [6]. These deletions are at least in part responsible for the lack of pathogenicity of these strains. Specific combinations of glycoproteins gene deletions in herpes simplex virus render the herpes simplex virus less pathogenic in mice [see 37 for review]. While, combinations of glycoprotein gene deletions in pseudorabies virus render the pseudorabies virus less pathogenic in swine [see 38 for review].
Herpesviruses contain non-essential regions of DNA in various parts of the genome, and that modification of these regions can attenuate the virus, leading to a non-pathogenic strain from which a vaccine may be derived. The degree of attenuation of the virus is important to the utility of the virus as a vaccine. Deletions which cause too much attenuation of the virus will result in a vaccine that fails to elicit an adequate immune response. Although several examples of attenuating deletions are known, the appropriate combination of deletions for any herpesvirus is not readily apparent.
Feline herpesvirus 1 (FHV) is the causative agent of feline viral rhinotracheitis, an acute upper respiratory disease in cats [7, 8]. Serological studies indicate that 50 to 70% of adult cats have detectable antibodies to the virus [9, 10]. Currently available inactivated and attenuated live virus vaccines reduce disease but do not prevent infection by FHV [11].
The feline herpesvirus is a member of the family herpesviridae, which are commonly known as the herpesviruses and a member of the subfamily alphaherpesvirus with a group D genome [12]. The FHV genome is comprised of approximately 134 kilobase (kb) pairs that is subdivided into a long unique segment of approximately 104 kb and a short unique segment of approximately 8 kb [13]. The unique short region is bounded by inverted repeat sequences which are approximately 11 kb. Physical maps of restriction endonuclease sites of the FHV genome have been published [12, 13]. The thymidine kinase gene of FHV has been sequenced and an FHV virus containing a deletion of the TK gene was isolated using a drug selection technique [14]. A feline herpesvirus having a deletion of the TK gene and an insertion of the feline leukemia virus (FeLV) envelope (env) and gag genes at the TK deletion site has been constructed [36]. Feline herpesviruses containing an insertion in the ORF1 and ORF2 region within the unique short [43], or a deletion in the gI and gE region of the unique short [44] have been described. A feline herpesvirus containing an insertion in a region of the unique long downstream of the FHV gC gene has been constructed [40,45].
The feline herpesviruses in this invention are useful as vectors for the delivery of vaccine antigens from microorganisms causing diseases in animals other than cats or dogs and for the delivery of therapeutic agents. The therapeutic agent that is delivered by a viral vector of the present invention must be a biological molecule that is a by-product of feline herpesvirus replication. This limits the therapeutic agent in the first analysis to either DNA, RNA or protein. There are examples of therapeutic agents from each of these classes of compounds in the form of antisense DNA, anti-sense RNA [18], ribozymes [19], suppressor tRNAs [20], interferon-inducing double stranded RNA and numerous examples of protein therapeutics, from hormones, e.g., insulin, to lymphokines, e.g., interferons and interleukins, to natural opiates. The discovery of these therapeutic agents and the elucidation of their structure and function does not necessarily allow one to use them in a viral vector delivery system
This invention provides a recombinant feline herpesvirus comprising a foreign DNA inserted into a feline herpesvirus genome, wherein the foreign DNA is inserted into a region of the genome which corresponds to a 3.0 kb EcoRI-SalI fragment within a SalI A fragment of the feline herpesvirus genome and is capable of being expressed in a host cell into which the virus is introduced.
Further this invention provides a recombinant feline herpesvirus comprising a feline herpesvirus genome, wherein the feline herpesvirus genome contains a deletion in a 3.0 kb EcoRI-SalI fragment of the SalI A fragment of the feline herpesvirus genome.
Lastly, this invention provides vaccines and methods of immunization of animals infected with feline herpesvirus.